An efficient chronic unpredictable stress protocol to induce stress-related responses in C57BL/6 mice

Abstract

Exposure to chronic stress can have broad effects on health ranging from increased predisposition for neuropsychiatric disorders to deregulation of immune responses. The chronic unpredictable stress (CUS) protocol has been widely used to study the impact of stress exposure in several animal models and consists in the random, intermittent, and unpredictable exposure to a variety of stressors during several weeks. CUS has consistently been shown to induce behavioral and immunological alterations typical of the chronic stress-response. Unfortunately C57BL/6 mice, one of the most widely used mouse strains, due to the great variety of genetically modified lines, seem to be resistant to the commonly used 4-week-long CUS protocol. The definition of an alternative CUS protocol allowing the use of C57BL/6 mice in chronic stress experiments is a need. Here, we show that by extending the CUS protocol to 8 weeks is possible to induce a chronic stress-response in C57BL/6 mice, as revealed by abrogated body weight gain, increased adrenals weight, and an overactive hypothalamic-pituitary-adrenal axis with increased levels of serum corticosterone. Moreover, we also observed stress-associated behavioral alterations, including the potentiation of anxious-like and depressive-like behaviors and a reduction of exploratory behavior, as well as subtle stress-related changes in the cell population of the thymus and of the spleen. The present protocol for C57BL/6 mice consistently triggers the spectrum of CUS-induced changes observed in rats and, thus, will be highly useful to researchers that need to use this particular mouse strain as an animal model of neuropsychiatric disorders and/or immune deregulation related to CUS.

Keywords: CUS; anxiety; chronic stress; depressive-like behavior; immune dysfunction; neuropsychiatric disorders; social defeat.

Figure 1

Effect of 4 weeks vs. 8 weeks of CUS on biometric parameters. Body weight gain for animals submitted to 4 (A) and 8 (B) weeks of CUS. Thymus weight and cellularity after exposure to 4 (C) and 8 (D) weeks of CUS. Adrenals weight after 4 (E) and 8 weeks of CUS (F). Corticosterone levels in the serum of animals from the group submitted to 4 (G) and 8 (H) weeks of CUS. Each bar/point represents the mean ± SEM from 10 animals per group. **p < 0.01, ***p < 0.001.

Figure 2

Impact of 4 and 8 weeks of CUS on anxious-like and locomotor behavior and exploratory activity. Behavioral performance of mice exposed to 4 (A) and to 8 weeks of CUS (B) in the EPM. Locomotor function of mice submitted to 4 (C) and 8 weeks (D) of CUS measured in the OF. Exploratory activity of mice submitted to 4 (E) and 8 weeks (F) of CUS measured in the OF. Each bar represents the mean ± SEM from 8 to 10 animals per group. *p < 0.05.

Figure 3

Impact of 4 weeks vs. 8 weeks of CUS on depressive-like behavior. Behavioral performance of mice submitted to 4 (A) and 8 weeks of CUS (B) in the FST. Behavioral performance of mice submitted to 4 (C) and 8 weeks of CUS in the TST (D). Each bar represents the mean ± SEM from 9 to 10 animals per group. *p < 0.05, **p < 0.01.

Figure 4

Impact of 8 weeks of CUS on cognition. Behavioral performance of mice exposed to 8 weeks of CUS in the MWM. Each point represents the mean ± SEM from 10 animals per group.

Figure 5

Impact of 4 weeks vs. 8 weeks of CUS on thymocyte subsets. Percentage of main cell populations in thymus after exposure to 4 (A) and 8 (B) weeks of CUS. Flow cytometry plot showing the gating strategy for thymocyte subsets (gate) (C). DN, double-negative thymocytes; DP, double-positive thymocytes; and SPCD4 and SPCD8, single-positive CD4 and CD8 thymocytes, respectively. Each bar represents the mean ± SEM from 10 animals per group. *p < 0.05.

Figure 6

Impact of 4 vs. 8 weeks of CUS on lymphoid cellular populations in the spleen. Percentage of T and B cells in spleen after exposure to 4 (A) and 8 (B) weeks of CUS. Percentage of CD4⁺ and CD8⁺ T cells in spleen after exposure to 4 (C) and 8 (D) weeks of CUS. Flow cytometry plot showing the gating strategy for T and B-lymphocytes (gates) (E). Each bar represents the mean ± SEM from eight animals per group.

Figure 7

Impact of 4 vs. 8 weeks of CUS on myeloid cellular populations in the spleen. Percentage of eosinophils, macrophages/dendritic cells, and neutrophils in spleen after exposure to 4 weeks (A) and 8 (B) weeks of CUS. Flow cytometry plot showing the gating strategy of myeloid splenocytes subsets (gates) (C). Each bar represents the mean ± SEM from 8 animals per group. *p < 0.05, **p < 0.01.